ES2740248T3 - Lipid nanoparticle compositions and methods for mRNA administration - Google Patents

Abstract

A composition comprising (a) at least one mRNA molecule at least a portion of which encodes a functional secreted polypeptide; and (b) a liposomal transfer vehicle comprising a lipid nanoparticle comprising one or more cationic lipids, one or more non-cationic lipids and one or more PEG modified lipids to use a method of treating a subject having a defect or deficiency in said secreted polypeptide, in which the mRNA is encapsulated in the transfer vehicle and in the composition is administered by pulmonary administration by nebulization.

Description

DESCRIPTION

Lipid nanoparticle compositions and methods for mRNA administration

[0001] New knowledge and therapies are still necessary for the treatment of protein and enzyme deficiencies. For example, storage diseases are a group of approximately 50 rare inherited metabolic disorders that in their lysosomal function, usually due to a deficiency of an enzyme required for metabolism. Fabry's disease is a lysosomal storage disease that results from the search for a deficiency of the alpha galactosidase enzyme (GLA), which causes a glycolipid known as globotriaosylceramide to accumulate in blood vessels and other tissues, leading to several painful manifestations For certain diseases, such as Fabry's disease, there is a need to replace a protein or enzyme that is normally secreted from cells to the bloodstream. Therapies, such as gene therapy, increase the level of production of a protein or enzyme. However, there have been several limitations to using conventional gene therapy for this purpose.

[0002] Conventional gene therapy involves the use of DNA for insertion of desired genetic information into host cells. The DNA in the cell generally integrates to some extent in the genome of one or more transfected cells, which allows for a lasting action of the genetic material in the host. While it has substantial benefits for such sustained action, the integration of exogenous DNA into the host genome can also have many harmful effects. For example, it is possible for DNA to be inserted into an intact gene, resulting in a mutation that prevents the function of the endogenous gene from being completely eliminated. Therefore, gene therapy with DNA can result in the performance of a vital genetic function in the treated treatment, such as the function of a delimited essential enzyme or the disruption of a gene critical for the regulation of cell growth, resulting in unregulated regulation or proliferation of cancer cells. Furthermore, with DNA-based gene therapy it is also possible that DNA-based genetic material leads to the induction of unwanted anti-DNA antibodies, which in turn can trigger a possibly fatal immune response. The effects of gene therapy that viral employees are also useful in an adverse immune response. In some circumstances, the viral vector may even integrate into the host genome. In addition, the production of clinical grade viral vectors is also expensive and time-consuming. It can also be difficult to control the distribution of genetic material. Therefore, although DNA-based gene therapy has been evaluated for the supply of secreted proteins using viral vectors (US Patent No. 6,066,626; US2004 / 0110709), these have been limited by these reasons.

[0003] Another apparent obstacle in these earlier principles in the delivery of nucleic acids encoding secreted proteins, that is, in the levels of protein that are ultimately produced. It is difficult to achieve significant levels of the desired protein in the blood and the amounts are not maintained over time. For example, the amount of protein produced by the nucleic acid supply does not reach normal physiological levels. See for example, US2004 / 0110709.

[0004] In contrast to DNA, the use of RNA as a genetic therapy agent is substantially safer because (1) RNA does not imply the risk of being stably integrated into the genome of the transfected cell, thus eliminating the concern of that the genetic release, the material, the normal functioning of an essential gene, caused a mutation that results in harmful or oncogenic effects; (2) foreign promoter sequences are not required for the translation of the encoded protein, avoiding new possible harmful effects; (3) a difference from plasmid DNA (pDNA), messenger RNA (mRNA) lacks CpG immunogenic motifs, so anti-RNA antibodies have not been generated; and (4) any detrimental effect resulting from RNA based on gene therapy would be limited to a relatively short half-life. In addition, it is not necessary for RNA to enter the nucleus to perform its function, while DNA must overcome this main barrier.

[0005] One reason why mRNA-based therapy has been used only in the past is that RNA is much less stable than DNA, especially when a cell's cytoplasm is reached and a degrading enzyme is exposed. The presence of a hydroxyl group in the second carbon of the sugar portion in the RNA is an impediment for the mRNA to form the structure of the double helix more stable in the DNA and, therefore, makes the mRNA more prone to hydrolytic degradation. As a result, until recently, mRNA is believed to be too important for transfection protocols. Advances in RNA stabilization modifications have aroused more interest in the use of mRNA in the place of plasmid DNA in gene therapy. Certain administration vehicles, such as cationic lipids or polymer administration vehicles can also help protect transfected mRNA from endogenous RNases. However, despite the increased stability of the modified mRNA, the delivery of mRNA to live cells in a manner that allows therapeutic levels of protein production remains a challenge, particularly for mRNA encoding full-length proteins. While the supply of mRNA encoding secreted proteins has been contemplated (US2009 / 0286852), the levels of a full-length secreted protein that will actually be produced through live mRNA delivery are not known and there is no reason to expect levels to exceed levels observed with DNA-based gene therapy.

[0006] To date, only significant progress has been made using mRNA gene therapy in applications For low translation levels it has not been a limiting factor, such as immunization with mRNA encoding antigens. Clinical trials involving vaccination against tumor antigens by intradermal injection of naked mRNA or with the protamine complex have demonstrated viability, lack of toxicity and promising results. X. Su et al., Mol. Pharmaceutics 8: 774-787 (2011). Unfortunately, low levels of translation have greatly restricted the exploitation of mRNA-based gene therapy in other applications that require higher levels of sustained expression of the protein encoded in mRNA to exert a biological or therapeutic effect.

[0007] WO 2007/024708 describes RNA molecules, oligoribonucleotides and polybibonucleotides comprising pseudofirin or a modified nucleoside.

[0008] Su et al. (Mol Pharm, June 6, 2011; 8 (3): 774-87) describes biodegradable nucleoshell structured nanoparticles with a poly (p-amino ester) (PBAE) core wrapped by a phospholipid bilayer shell for delivery of mRNA-based vaccines.

[0009] Kormann et al. (Nat Biotechnol. 2011 Feb; 29 (2): 154-7) investigates the therapeutic utility of chemically modified RNA as an alternative to DNA-based gene therapy.

[0010] WO 2011/068810 describes the compositions and methods for modulating the expression of a gene or the production of a protein by transfecting the target cells with nucleic acids.

[0011] US 2012/0195936 discloses a polyribonucleotide with a sequence encoding a protein or protein fragment, and the polyiribonucleotide a combination of unmodified and modified nucleotides.

[0012] The invention of a composition comprising (a) at least one mRNA molecule at least a part of which encodes a functional secreted polypeptide; and (b) a liposomal transfer vehicle comprising a lipid nanoparticle comprising one or more cationic lipids, one or more non-cationic lipids and one or more lipids modified with PEG to use a method of treating a subject having a defect or deficiency in said secreted polypeptide, in which the mRNA is encapsulated in the transfer vehicle and in the composition is administered by pulmonary administration by nebulization.

[0013] Administration of mRNA gene therapeutic agents according to the invention leads to the production of effective therapeutic levels of secreted proteins through a "deposition effect". The mRNA encoding a secret protein is loaded into lipid nanoparticles and administered to the target cells in vivo. The target cells then act as a source of deposit for the production of soluble and secreted proteins in the circulatory system at therapeutic levels. In some places, protein levels.

[0014] The compositions of the invention are useful in the management and treatment of a large number of diseases, in particular protein diseases and / or enzyme deficiencies, in which the protein or enzyme is normally secreted. Individuals suffering from the diseases may have underlying genetic defects that lead to the compromised expression of a protein or enzyme, including, for example, the non-synthesis of the secreted protein, the synthesis of the secreted protein or the synthesis of a secreted protein that It lacks biological activity or has it to a lesser extent.

[0015] The mRNA can encode a clinically useful secreted protein. For example, mRNA can encode a functional secreted urea cycle enzyme or a secreted enzyme involved in lysosomal storage disorders. The mRNA can encode, for example, erythropoietin (for example, human EPO) or a-galactosidase (for example, human a-galactosidase (human GLA).

[0016] In some embodiments of the invention, the mRNA may comprise one or more modifications that provide stability to the mRNA (for example, as compared to a wild type or a native version of the mRNA) and may also comprise one or more modifications related to the wild type that correct an implicit defect in the aberrant expression associated with the protein. For example, the nucleic acids of the present invention may be modifications in one or both of the 5 'and 3' untranslated regions. Such modifications may include a partial sequence of an immediate-early cytomegalovirus (CMV) 1 (IE1) gene, a poly A tail, a Cap1 structure or a sequence encoding human growth hormone (hGH)). In some parts, mRNA is modified to decrease mRNA immunogeneity.

[0017] Methods of treating a subject comprising a composition of the invention are also contemplated. For example, the methods for treating and preventing conditions in the production of a particular secreted protein and / or the use of a secret protein is inappropriate or compromised. The methods included in this document can be used to treat a subject that has a deficiency in one or more enzymes of the urea cycle or in one or more enzymes deficient in a lysosomal storage disorder.

[0018] The mRNA in the compositions of the invention is formulated in a liposomal transfer vehicle to facilitate delivery to the target cell. Transfer vehicles of one or more cationic lipids, non-lipids cationic and lipid modified with PEG. For example, the transfer vehicle may comprise at least one of the following cationic articles: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000 or HGT5001. At the site, the transfer vehicle comprises cholesterol (col) and / or a PEG modified lipid. On some occasions, DMG-PEG2K transfer vehicles. In certain parts, the transfer vehicle comprises one of the following lipid formulations: C12-200, DOPE, col, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, col, DMG-PEG2K, HGT5001, DOPE, col, DMG-PEG2K.

[0019] In embodiments of the invention, the secreted protein is produced by the target cell for sustained amounts of time. For example, the secreted protein can be produced for more than one hour, more than four, more than six, more than 24, more than 48 hours or more than 72 hours after administration. In some parts, the polypeptide is expressed at a maximum level approximately six hours after administration. In some parts, polypeptide expression is maintained at least at a therapeutic level. In some responses, the polypeptide is expressed in less a therapeutic level in more than one, more than four, more than 12, more than 24, more than 48 hours or more than 72 hours after administration. In some parts, the polypeptide is detectable at the patient's serum or tissue level (for example, liver or lung). In some responses, the level of detectable polypeptide is the result of continuous expression of the mRNA composition for periods of time of more than one, more than four, more than 12, more than 24, more than 48 hours or more than 72 hours after administration.

[0020] In certain parts, the secreted protein produces levels higher than normal physiological levels. The level of secreted protein may increase compared to a control.

[0021] In some parts, control is the baseline physiological level of the polypeptide in a normal individual or in a population of normal individuals. Elsewhere, control is the physiological level of the polypeptide reference in an individual that has a deficiency in the relevant protein or polypeptide or in a population of individuals that has a deficiency in the relevant protein or polypeptide. In some parts, the control may be the normal level of the relevant protein or polypeptide in the individual to whom the composition is administered. Elsewhere, the control is the level of expression of the polypeptide in another therapeutic intervention, for example, in the direct injection of the corresponding polypeptide, in one or more comparable time points.

[0022] In certain parts, the polypeptide is expressed by the target cell at a level that is at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, 30 times, at least 100 times, at least 500 times, at least 5000 times, at least 50,000 times or at least 100,000 times greater than a control. Later, more than 12, more than 24, or more than 48 hours, or more than 72 hours after administration. For example, in one embodiment, protein levels are detected in the serum at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, 30 times, at least 100 times , at least 500 times, at least 5000 times, at least 50,000 times or at least 100,000 times more than a control for at least 48 hours or 2 days. In certain names, protein levels are detectable at 3 days, 4 days, 5 days, or 1 week or more after administration. Increased levels of secreted protein can be observed in the serum and / or in a tissue (for example, liver, lung).

[0023] In some embodiments, the described method produces a sustained half-life of the desired secreted protein. For example, the secreted protein can be detected for hours or days longer than the half-life observed through subcutaneous injection of the secreted protein. In place, the half-life of the protein is maintained for more than 1 day, 2 days, 3 days, 4 days, 5 days, or 1 week or more.

[0024] In some of the parts of the invention, administration is applied to repeated ones.

[0025] The polypeptide can be, for example, one or more of erythropoietin, a-galactosidase, the LDL receptor, factor VIII, factor IX, aL-iduronidase (for MPS I), sulfatase iduronate (for MPS II), heparin -N-sulfatase (for MPS IIIA), aN-acetylglucosaminidase (for MPS IIIB), galactose 6-sultatase (for MPS IVA), lysosomal acid lipase, arylsulfatase-A.

[0026] The invention relates to a composition that provides mRNA to a cell or subject, at least part of which encodes a functional protein, in an amount that is substantially less than the amount of the corresponding functional protein generated from that mRNA. In other words, in certain embodiments, the mRNA administered to the cell can produce an amount of protein that is substantially greater than the amount of mRNA administered to the cell. For example, in a given amount of time, for example 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or 24 hours from the administration of the RNA to a cell or subject , the amount of corresponding protein generated by that mRNA can be at least 1.5, 2, 3, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 400, 500 or more times greater than the amount of mRNA actually administered to the cell or the subject. This can be measured on a mass per mass basis, on a mole basis per mole, and / or on a molecule basis per molecule. Protein can be measured in several ways. For example, for a cell, the measured protein can be measured as intracellular protein, extracellular protein or a combination of the two. For a subject, the measured protein may be whey measured protein; in a specific tissue or in tissues such as the liver, kidney, heart or brain; in a specific cell type, such as one of several types of liver or brain cells; or in any combination of serum, tissue and / or cell type. In addition, a reference amount of the endogenous protein in the cell or one person before mRNA administration and then subtracting the measured protein after mRNA administration. In this way, the RNA can provide a source of deposit or a large amount of therapeutic material to the cell or to the subject, for example, compared to the amount of RNA administered to the cell or to the subject. The deposit source can act as a continuous source for mRNA polypeptide expression for sustained periods of time.

[0027] The other features were discussed and the concomitant advantages and advantages of the present invention will be better understood by the following detailed description of the invention when taken in conjunction with the accompanying examples. The various publications in this document are complementary and the meetings may be combined or used in a manner understood by the person skilled in the art in view of the teachings contained in this document.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]

FIG. 1 Sample of the nucleotide sequence of a 5 'CMV sequence (SEQ ID NO: 1), in which X, if present, is GGA.

FIG. 2 Shows the nucleotide sequence of a 3 'hGH sequence (SEQ ID NO: 2).

FIG. 3 Shows the nucleotide sequence of human erythropoietic mRNA (EPO) (SEQ ID NO: 3). This sequence may be flanked at the 5 'end with SEQ ID NO: 1 and at the 3' end with SEQ ID NO: 2.

FIG. 4 Shows the nucleotide sequence of the human alpha-galactosidase (GLA) mRNA (SEQ ID NO: 4). This sequence may be flanked at the 5 'end with SEQ ID NO: 1 and at the 3' end with SEQ ID NO: 2.

FIG. 5 Shows the nucleotide sequence of the human alpha-1 antitrypsin (A1AT) mRNA (SEQ ID NO: 5). This sequence may be flanked at the 5 'end with SEQ ID NO: 1 and at the 3' end with SEQ ID NO: 2.

FIG. 6 Sample nucleotide sequence of human factor IX mRNA (FIX) (SEQ ID NO: 6). This sequence may be flanked at the 5 'end with SEQ ID NO: 1 and at the 3' end with SEQ ID NO: 2.

FIG. 7 shows the quantification of secreted hEPO protein levels measured through ELISA. The protein detected is the result of its production from hEPO mRNA administered intravenously through a single dose of various lipid nanoparticle formulations. The formulations C12-200 (30 ug), HGT4003 (150 ug), ICE (100 ug), DODAP (200 ug) are considered as the cationic / ionizable lipid component of each test article ( Formulations 1-4). Values are based on a blood sample four hours after administration.

FIG. 8 Shows the hematocrit measurement of mice with a single IV dose of lipid nanoparticles loaded with human EPO mRNA ( Formulations 1-4). Samples of whole blood were taken at 4 hours (day 1), 24 hours (day 2), 4 days, 7 days and 10 days after administration.

FIG. 9 Shows hematocrit measurements of mice with lipid nanoparticles loaded with human EPO-mRNA, either with a single IV dose or three injections (day 1, day 3, day 5). Blood samples were taken before injection (day -4), day 7 and day 15. Formulation 1 was administered : (30 ug, single dose) or (3 x 10 ug, dose day 1, day 3, day 5 ); Formulation 2 was administered: (3 x 50 ug, dose day 1, day 3, day 5). FIG. 10 shows the quantification of levels of secreted human a-galactosidase protein (hGLA), measured through ELISA. The protein detected is the result of the production of hGLA mRNA administered through lipid nanoparticles ( Formulation 1; 30 ug single intravenous dose, based on encapsulated mRNA). HGLA protein is detected through 48 hours.

FIG. 11 Shows serum hGLA activity. HGLA activity was measured using the 4-methylumberliferyl-aD-galactopyranoside (4-MU-a-gal) substrate at 37 ° C. The data is an average of 6 to 9 individual measurements.

FIG. 12 shows the quantification of serum hGLA protein levels measured through ELISA. Protein is produced from hGLA mRNA administered through lipid nanoparticles based on C12-200- (C12-200: DOPE: Chol: DMGPEG2K, 40: 30: 25: 5 ( formulation 1); 30 gg based mRNA in encapsulated mRNA, single dose IV). The hGLA protein is controlled for 72 hours. By single intravenous dose, based on encapsulated mRNA). The hGLA protein is controlled for 72 hours.

FIG. 13 shows the quantification of hGLA protein levels in liver, kidney and spleen, measured through ELISA. The protein is produced from hGLA mRNA administered through lipid nanoparticles based on C12-200 ( Formulation 1; 30 gg of mRNA based on encapsulated mRNA, single dose IV). The hGLA protein is controlled for 72 hours.

FIG. 14 shows a dose response study that monitors the production of hGLA proteins as human GLA protein derived from MRT secreted in serum (A) and liver (B). Samples were measured 24 hours after administration ( Formulation 1; single IV dose, N = 4 mice / group) and quantified by ELISA.

FIG. 15 shows the pharmacokinetic profiles of alpha-galactosidase based on ERT in athymic mice (40 ug / kg dose) and hGLA protein produced from MRT ( Formulation 1; 1.0 mg / kg mRNA dose).

FIG. 16 shows the quantification of hGLA protein levels secreted in mice. The hGLA protein is produced from hGLA mRNA administered through C12-200-based lipid nanoparticles ( Formulation 1; 10 pg of mRNA per single intravenous dose, according to the encapsulated mRNA). The serum is controlled for 72 hours.

FIG. 17 shows the quantification of hGLA protein levels in the liver, kidney, spleen and heart of Fabry KO mice treated with MRT, measured through ELISA. The protein is produced from hGLA mRNA administered through lipid nanoparticles based on C12-200 ( Formulation 1; 30 pg of mRNA based on encapsulated mRNA, single dose IV). The hGLA protein is controlled for 72 hours. The literature values that represent normal physiological levels are plotted as dashed lines. FIG. 18 Shows the quantification of hGLA protein levels secreted in MRT and in Fabry mice treated with alpha-galactosidase, according to the ELISA method. Both therapies were administered as a single intravenous dose of 1.0 mg / kg.

FIG. 19 shows the quantification of hGLA protein levels in liver, kidney, spleen and heart of Fabry KO mice treated with MRT and ERT (alpha-galactosidase) as measured by ELISA. Protein produced from hGLA mRNA administered through lipid nanoparticles ( Formulation 1; 1.0 mg / kg mRNA based on encapsulated mRNA, single dose IV).

FIG. 20 Shows the relative quantification of globotrioasilceramide (Gb3) and lyso-Gb3 in the kidneys of treated and untreated mice. Fabry KO male mice were treated with a single dose of lipid nanoparticles loaded with GLA mRNA or alpha-galactosidase at 1.0 mg / kg. The amount of Gb3 / smooth-Gb3 one week after administration.

FIG. 21 Shows the relative quantification of globotrioasilceramide (Gb3) and lyso-Gb3 in the heart of treated and untreated mice. Fabry KO male mice were treated with a single dose of lipid nanoparticles loaded with GLA mRNA or alpha-galactosidase at 1.0 mg / kg. The amount of Gb3 / smooth-Gb3 one week after administration.

FIG. 22 It shows a dose response study that monitors the production of GLA protein as human GLA protein derived from serum secreted MRT. Samples were measured 24 hours after administration (single dose, IV, N = 4 mice / group) of HGT4003 ( Formulation 3) or of lipid nanoparticles based on HGT5000 ( Formulation 5) and quantified by ELISA.

FIG. 23 sample of hGLA protein production measured in serum (A) or in liver, kidney and spleen (B). Samples were measured 6 hours and 24 hours after administration (single dose, IV, N = 4 mice / group) of lipid nanoparticles based on HGT5001 ( Formulation 6) and quantified by ELISA.

FIG. 24 shows the quantification of secreted human factor IX protein levels measured using ELISA (mean standard deviation ng / mL ±). The FIX protein is produced from FIX mRNA administered through lipid nanoparticles based on C12-200 (C12-200: DOPE: Chol: DMGPEG2K, 40: 30: 25: 5 ( Formulation 1); 30 pg of mRNA per single intravenous dose, based on encapsulated mRNA). The FIX protein is controlled through 72 hours. (n = 24 mice)

FIG. 25 shows the quantification of levels of secreted human α-1-antitrypsin protein (A1AT) measured by ELISA. The A1AT protein is produced from A1AT mRNA administered through lipid nanoparticles based on C12-200 (C12-200: DOPE: Chol: DMGPEG2K, 40: 30: 25: 5 ( Formulation 1); 30 pg of mRNA per dose intravenous single, based on encapsulated mRNA). The A1AT protein is controlled for 24 hours.

FIG. 26 shows an ELISA-based quantification of hEPO protein detected in the lungs and serum of mice after intratracheal administration of hEPO mRNA-loaded nanoparticles (measured mIU) (C12-200, HGT5000 or lipid nanoparticles and in HGT5001; Formulations 1, 5, 6, respectively). Animals were sacrificed 6 hours after administration (n = 4 mice per group).

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0029] The description of the functions and methods for intracellular administration of mRNA in a liposomal transfer vehicle to one or more target cells for the production of therapeutic levels of the secreted functional protein.

[0030] The term "functional", as used herein to qualify a protein or enzyme, means that the protein or enzyme has biological activity, or, alternatively, is capable of performing the same, or a function similar to the protein or native or normal functioning enzyme. The mRNA compositions of the invention are useful for the treatment of various metabolic or genetic disorders, and in particular those genetic or metabolic that involve non-expression, erroneous expression or deficiency of a protein or enzyme. The term "therapeutic levels" refers to the levels of the protein detected in the blood or tissues that are at the top of the control levels, where the control can be normal physiological levels, or the levels in the subject before administration of the mRNA composition. The term "secreted" refers to the protein that is detected outside the target cell, in the extracellular space. Protein can be detected in the blood or tissues. In the context of the present invention, the term "produced" is used in its broadest sense for the translation of less to an mRNA into a protein or enzyme. As noted herein, the parts include a transfer vehicle. As used herein, the term "transfer vehicle" includes any of the pharmaceutical vehicles, diluents, excipients and the like found in relation to the administration of biologically active agents, including nucleic acids. The compositions and, in particular, the transfer vehicles herein are capable of providing mRNA to the target cell.

MRNA

[0031] The mRNA in the compositions of the invention can encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide or other protein of interest that is normally secreted. In one embodiment of the invention, the RNA may have the functionality of biological techniques that, for example, improve the stability and / or the half-life of said mRNA that improve it in protein production.

[0032] One or more unique mRNAs can be co-administered to target cells, for example, by combining two unique mRNAs is a single transfer vehicle. In one embodiment of the present invention, a therapeutic mRNA primer, and a second therapeutic mRNA, can be formulated in a single transfer and administration vehicle. The present invention also contemplates the joint administration and / or the co-administration of a therapeutic mRNA primer and a second nucleic acid to facilitate and / or improve the function or delivery of the therapeutic mRNA primer. For example, said second nucleic acid (for example, exogenous or synthetic mRNA) can encode a membrane transporter protein which, when expressed (eg, translation of exogenous or synthetic mRNA) facilitates the delivery or improvement of the biological activity of the primer MRNA Alternatively, the therapeutic mRNA primer can be administered with a second nucleic acid that functions as a "chaperone", for example, to direct the folding of any of the first therapeutic mRNAs.

[0033] Methods are also contemplated that allow the administration of one or more therapeutic nucleic acids for the treatment of a single disorder or deficiency, where each therapeutic nucleic acid functions as a different mechanism of action. For example, the compositions of the present invention may comprise a therapeutic mRNA primer that, for example, is administered to correct a deficiency of endogenous enzymes or proteins, and which is accompanied by a second nucleic acid, which can be administered to deactivate or " knock-down "an endogenous malfunctioning nucleic acid and its protein or enzyme product. Such" second "nucleic acids may encode, for example, mRNA or siRNA.

[0034] After transfection, a natural mRNA in the compositions of the invention can decay with a half-life of between 30 minutes and several days. The mRNA in the compositions of the invention translates into a smaller amount of ability to be translated, thus producing a functional secreted protein or enzyme. Therefore, the invention has been stabilized. In some activities of the invention, mRNA activity is prolonged for a prolonged period of time. For example, the activity of the mRNA can be prolonged such that the compositions of the present publication are administered weekly or biweekly, or more preferably monthly, bi-monthly, quarterly or annually. The activity is prolonged over time. Similarly, the activity of the compositions of the present invention can be extended or further extended by modifying the power of the translation system. In addition, the amount of protein or functional enzyme produced by the target cell is a function of the amount of mRNA administered to the target cells and the stability of said mRNA. To the extent that the mRNA stability of the present invention can improve or increase the half-life, the activity of the secreted protein or enzyme produced and the dosage frequency of the composition can be further extended.

[0035] Accordingly, in some embodiments, the mRNA in the compositions of the invention comprise at least one modification that grants increased or improved stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms "modification" and "modified" as such terms relate to the nucleic acids provided herein, include at least one alteration that improves stability and makes mRNA more stable (eg. , resistant to nuclease digestion As used herein, the terms "stable" and "stability" refer to the nucleic acids of the present invention, and, in particular, with respect to mRNA, refer to a increased or improved resistance to degradation by, for example, nucleases (i.e. endonucleases or exonucleases) that are normally capable of degrading said mRNA. Increased stability may include, for example, less sensitivity to hydrolysis or other destruction by part of the endogenous enzymes (for example, endonucleases or exonucleases) or conditions within the cell or target tissue, increase or improve the resistance of said mRNA in the cell, target tissue, subject and / or cytoplasm. The mRNA variables are improved in this document. Also contemplated by the terms "modification" and "modified", to the extent that such terms relate to the mRNA of the present invention, there are alterations that increase or improve the translation of the mRNA nucleic acids, including, for example, the inclusion of sequences that work at the beginning of protein translation (for example, the Kozac consensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

[0036] In some parts, the mRNAs of the invention have been a chemical or biological way to make them more stable. Modifications of an RNA include the depletion of a base or the modification of a base, for example, the chemical modification of a base. The phrase "chemical modifications", as used herein, includes modifications that occur in natural mRNA, for example, covalent modifications, such as the introduction of modified nucleotides (eg, nucleotide analogs or the inclusion of pending groups ) that are not found naturally in such mRNA molecules).

[0037] In addition, the modifications are adapted to one or more nucleotides of a code so that the code encodes the same amino acid but is more stable than the code found in the wild-type version of the mRNA. For example, an inverse relationship between RNA stability and a greater number of cytidines (C) and / or uridine residues (U) has been demonstrated, and RNA without residues of C and U has been found to be stable for Most RNases (Heidenreich, et al., J Biol Chem 269, 2131-8 (1994)). On some occasions, the number of residues C and / or U in an mRNA sequence is reduced. In another embodiment, the number of residues C and / or U is reduced by replacing a code encoding a particular amino acid another code encoding the same amino acid or a related amino acid. The modifications contemplated in the mRNA nucleic acids of the present invention also include the incorporation of pseudouridines. The incorporation of pseudouridines into the nucleic acids of the present invention can improve stability and translation capacity, as well as decrease immunogenicity. Live. See, for example, Karikó, K., et al., Molecular Therapy 16 (11): 1833-1840 (2008). Substitutions and modifications herein may be made by methods readily known to one skilled in the art.

[0038] The limitations in reducing the number of residues of C and U in a sequence will probably be greater in the coding region of an mRNA, in comparison with an untranslated region, (ie, it is probably not possible eliminate all residues C and U present in the message while retaining the ability of the message to encode the desired amino acid sequence). However, the degeneracy of the genetic code presents an opportunity to allow the number of C and / or residues that are present in the sequence to be reduced, while maintaining the same coding capacity (ie, depending on the amino acid being encoded by a codon, several different possibilities for the modification of RNA sequences may be possible). For example, codons for Gly can be modified to GGA or GGG instead of GGU or GGC.

[0039] The term modification also includes, for example, the incorporation of non-nucleotide or modified nucleotide bonds into the mRNA sequences of the present invention (for example, modifications of one or both 3 'and 5' ends of a molecule of MRNA encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer polyA tail), the alteration of the 3 'UTR or the 5' UTR, complexing the mRNA with an agent (for example, a protein or a complementary nucleic acid molecule), and inclusion of elements that change the structure of an mRNA molecule (for example, that form secondary structures).

[0040] Poly A tail is thought to stabilize natural messengers. Therefore, in one embodiment, a long tail of poly A can be added to an mRNA molecule, which makes the mRNA more stable. Poly A tails can be added using a variety of techniques recognized in the art. For example, long poly A tail can be contacted with synthetic or in vitro transcribed mRNAs using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from the PCR products. In one embodiment, the length of the poly A tail is at least about 90, 200, 300, 400 at least 500 nucleotides. In one embodiment, the length of the poly A tail is adjusted to control the stability of a modified RNA molecule of the invention and, therefore, protein transcription. For example, since the length of the poly A tail can influence the half-life of an mRNA molecule, the length of the poly A tail can be adjusted to modify the level of mRNA resistance when controlling the course of protein expression time in a cell. In one embodiment, the stabilized mRNA molecules are sufficiently resistant to degradation in vivo (for example, by nucleases), so that they can be administered to the target cell without a transfer vehicle.

[0041] In one embodiment, an mRNA can be modified by incorporating 3 'and / or 5' untranslated sequences (UTR) that are not naturally found in the wild-type mRNA. In one embodiment, the 3 'and / or 5' flanking sequence that naturally flanks an mRNA and encodes a second protein cannot be incorporated into the nucleotide sequence of an mRNA molecule that encodes a therapeutic or functional protein to modify it. For example, the 3 'or 5' sequences of mRNA molecules that are stable (eg, globin, actin, GAPDH, tubulin, histone or citric acid cycle enzymes) can be incorporated into the 3 'and / or 5 region 'of a sense mRNA nucleic acid molecule to increase the stability of the sense mRNA molecule. See, for example, US2003 / 0083272.

[0042] In some embodiments, the mRNA in the compositions of the invention includes modification of the 5 'end of the mRNA to include a partial sequence of the immediate-early CMV 1 (IE1) 1 (IE1) gene, or a fragment thereof. (for example, SEQ ID NO: 1) to improve nuclease resistance and / or improve mRNA half-life. In addition to increasing the stability of the mRNA nucleic acid sequence, the inclusion of a partial CMV early gene sequence 1 (IE1) and the expression of the functional protein or enzyme has been discovered and surprised. Also included is the inclusion of a sequence of the human growth gene (hGH), or a fragment thereof (for example, SEQ ID NO: 2) at the 3 'ends of the nucleic acid (eg, mRNA) to stabilize even more mRNA. Generally, preferred modifications improve the stability and / or pharmacokinetic properties (eg, half-life) of mRNA in relation to its unmodified counterparts, and include, for example, modifications to improve the resistance of said mRNA to digestion of nucleases in vivo.

[0043] In addition, the variants of the nucleic acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, in which the functions of nucleic acids that include mRNA stabilization and / or pharmacokinetic properties (for example, half-life). Variants can have more than 90%, more than 95%, more than 98% or more than 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0044] In some embodiments, the composition may comprise a stabilizing reagent. The compositions can be included in one or more formulation reagents that bind directly or indirectly, and stabilize the mRNA, thereby improving residence time in the target cell. Such reagents preferably lead to an improved life of mRNA in the target cells. For example, the stability of an RNA and the translation efficiency are increased by the incorporation of "stabilizing reagents" that form complexes with the RNA that are naturally maintained within a cell (see, for example, US Pat. No. 5,677,124). The incorporation of a stabilizing reagent can be achieved, for example, by combining poly A and a protein with the mRNA for in vitro stabilization before loading or encapsulating the mRNA into a transfer vehicle. Stabilization reagents are integrated into one or more proteins, peptides, aptamers, accessory translation protein, mRNA binding proteins and / or translation initiation factors.

[0045] The stabilization of functions can also be improved by using the inhibitory moieties of the operation, which are suitable, for example, by interconnecting a lipid soluble anchor in the membrane itself, or by joining direct to membrane lipid groups). These hydrophilic polymers that inhibit opsonization form a protective layer that decreases liposome uptake of the macrophage monocyte, monocyte and endothelial reticulum system (for example, as described in US Patent No. 4,920,016). Modified transfer vehicles with operating inhibition residues remain in the circulation much longer than their unmodified counterparts.

[0046] When RNA is hybridized to a complementary nucleic acid molecule (eg DNA or RNA), it can be protected from nucleases. (Krieg, et al. Melton. Methods in Enzymology. 1987; 155, 397-415). The stability of the hybridized RNA is probably due to the inherent specificity of a single strand of most RNases. On some occasions, the stabilization reagent selected to complex an mRNA is a eukaryotic protein (for example, a mammalian protein). In yet another embodiment, the mRNA can be modified by hybridization with a second nucleic acid molecule. If a mRNA molecule hybridizes with a complementary nucleic acid molecule, translation initiation can be reduced. In some parts, the 5 'untranslated region and the AUG start region of the mRNA molecule can optionally be left unhybridized. After the initiation of translation, the development activity of the ribosome complex can function even in the high affinity duplex so that translation can continue. (Liebhaber. J. Mol. Biol. 1992; 226: 2-13; Monia, et al. J Biol Chem. 1993; 268: 14514-22.)

[0047] It will be understood that any of the methods has been used in combination with one or more of the other methods and / or can be used.

[0048] The mRNA of the present invention may optionally be combined with a reporter gene (for example, upstream or downstream of the mRNA coding region) which, for example, facilitates the determination of mRNA administration to cells or tissues. Diana. The genes may include, for example, green fluorescent protein mRNA, Renilla luciferase mRNA (luciferase mRNA), firefly luciferase mRNA, or combinations thereofd. For example, the GFP mRNA can be fused with an mRNA encoding a secreted protein to facilitate confirmation of the location of the mRNA in the target cells that acts as a reservoir for protein production.

[0049] The terms "transfect" or "transfection" refer to the intracellular introduction of an mRNA into a cell, or preferably into a target cell. The mRNA can become a stable or transient form in the target cell. The term "transfection efficiency" refers to the relative amount of mRNA captured by the target cell that is subject to a transfection. In practice, the efficiency of transfection is estimated by the amount of a reporter nucleic acid product expressed by the target cells after transfection. Preferred features are used with the same transfection efficiencies and, in particular, the adverse effects that occur by transfection of non-target cells are minimized. The responses of the target cell, while minimizing possible systemic adverse effects. In one embodiment of the present invention, the transfer vehicles of the present invention are the same as large mRNAs (eg, mRNA of at least 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 5kDa, 10kDa, 12kDa, 15kDa, 20kDa, 25kDa, 30kDa, or more). Liposomes as transfer vehicles of the invention encapsulate mRNA without compromising biological activity. In some embodiments, the transfer vehicle demonstrates a preferential and / or substantial binding to a target cell with respect to non-target cells. The transfer vehicle manages its content to the target cell so that the mRNA is sent to the appropriate subcellular compartment, such as the cytoplasm.

Transfer vehicle

[0050] The transfer vehicle in the compositions of the invention is a liposomal transfer vehicle comprising a lipid nanoparticle. The transfer vehicle can be selected and / or prepared to optimize the delivery of the mRNA to a target cell.

[0051] Liposomal transfer vehicles are used to facilitate the administration of nucleic acids to target cells. Liposomes (i.e., liposomal lipid nanoparticles) are generally useful in a variety of applications in research, industry and medicine, in particular for their use as transfer vehicles for diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16 : 307-321, 1998; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) and is characterized as microscopic vesicles that have an internal aquatic space of an external environment. By a membrane of one or more bilayers. The bilayer membranes of the liposomes are formed in a specific way, such as lipids of synthetic or natural origin that have spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). The membranes of the liposome layers can also be formed by amphiphilic polymers and surfactants (eg, polymeric, niosomes, etc.).

[0052] In the context of the present invention, a liposomal transfer vehicle serves to transport the mRNA to the target cell. For the purposes of the present invention, liposomal transfer vehicles are prepared to contain the desired nucleic acids. The process of incorporating a desired entity (eg, a nucleic acid) into a liposome is often referred to as "loading" (Lasic, et al., FEBS Lett., 312: 255-258, 1992). The incorporation of a nucleic acid into liposomes is also referred to herein as "encapsulation" in which the nucleic acid is completely within the interior space of the liposome. The purpose of incorporating an mRNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid or an environment that may have enzymes or chemicals that degrade nucleic acids and / or the systems or receptors that cause rapid excretion of nucleic acids. Therefore, the selected transfer vehicle is able to improve the stability of the mRNA contained therein. The liposome may allow the encapsulated mRNA to reach the target cell and / or may preferably allow the encapsulated mRNA to reach the target cell or, alternatively, limit the supply of said mRNA to other sites or cells where the presence of the administered mRNA may be useless. or undesirable. In addition, the incorporation of mRNA into a cationic liposome also facilitates the delivery of said mRNA into a target cell.

[0053] Ideally, liposomal transfer vehicles are prepared to encapsulate one or more desired mRNAs so that the compositions demonstrate high transfection efficiency and improved stability. While liposomes can facilitate the introduction of nucleic acids into target cells, the addition of polycations (for example, poly L-lysine and protamine), as a copolymer can facilitate, and in some cases remarkably enhances the efficiency of transfection of various types of cationic liposomes. 2-28 times in several cell lines both in vitro and in vivo. (See NJ Caplen, et al., Gene Ther. 1995; 2: 603; S. Li, et al., Gene Ther.

1997; 4, 891.)

Lipid Nanoparticles

[0054] In the present invention, the transfer vehicle is formulated as a lipid nanoparticle. As used herein, the phrase "lipid nanoparticle" refers to a transfer vehicle comprising one or more cationic lipids, non-cationic lipids and PEG modified lipids. Preferably, the lipid nanoparticles are formulated for delivery of one or more mRNAs to one or more target cells. Examples of suitable lipids include, for example, phosphatidyl compounds (for example, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides). In one embodiment, the transfer vehicle is selected based on the ability to facilitate the transfer of a target cell.

[0055] The invention contemplates the use of lipid nanoparticles as transfer vehicles comprising a cationic lipid to encapsulate and / or improve mRNA administration in the target cell that will act as a reservoir for protein production. As used herein, the phrase "cationic lipid" refers to any of a number of lipid species that carry a net positive charge and a selected pH, such as physiological pH. The lipid nanoparticles contemplated are prepared with mixtures of multi-component lipids of varying proportions using one or more cationic lipids, non-cationic lipids and PEG modified lipids. Several cationic lipids have been described in the literature, many of which are commercially available.

[0056] Cationic lipids particularly suitable for use in the compositions of the invention include those described in international patent publication WO 2010/053572, and more particularly, C12-200 described in paragraph [00225] of WO 2010/053572. In certain responses, the compositions of the invention employ lipid nanoparticles that provide an ionizable cationic lipid are described in the U.S. ^provisional patent application. ^{61 / 617,468, published March 29, 2012, for example, (15Z, 18Z) -N, N-dimethyl-6- (9Z, 12Z) -octadeca-9, 12-dien-1-yl) tetracosa-15 , 18-dien-1-amine (HGT5000), (15Z, 18Z) -N, N-dimethyl-6 - ((9Z, 1} 2z) -octadeca-9, 12-dien-1-yl) tetracosa-4, 15,18-trien-1-amine (HGT5001) and (15Z, 18Z) -N, N-dimethyl-6 - ((9Z, 12Z) -octadeca-9,12-dien-1-yl) tetracosa-5, 15,18-trien-1-amine (HGT5002).

[0057] In some parts, the cationic lipid chloride N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium or "DOTMA" is used. (Felgner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); US Patent No. 4,897,355). DOTMA may be formulated alone or may be combined with the neutral lipid, dioleoylphosphatidyl ethanolamine or " DOPE "or other cationic or non-cationic lipids in a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to improve the delivery of nucleic acids to the target cells. Other suitable cationic lipids they include, for example, 5-carboxy-spermylglycinadioctadecylamide or "DOGS", 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium or "DOSPA" (Behr et al. Proc Nat.'1 Acad. Sci 86, 6982 (1989); U.S. Patent No. 5,171,678; U.S. Patent No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium propane or "DODAP ", 1,2-dioleoyl-3-trimethylammonium propane or" DOTAP ". The cationic lipids contemplated also include 1,2-distearyloxy-N, N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N, N-dimethyl-3-aminopropane or "DODMA", 1,2- dilinoleyloxy -N, N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N, N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N, N-dimethylammonium chloride or "DODAC" , N, N-distearyl-N, N-dimethylammonium bromide or "DDAB", N- (1,2-dimiristyloxypropyl-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide or "DMRIE", 3- dimethylamino-2- (colest-5-en-3-beta-oxibutan-4-oxi) -1- (cis, cis-9,12-octadecadienoxy) propane or "CLinDMA", 2- [5 '- (colest- 5-en-3-betaoxy) -3'-oxapentoxy) -3-dimethyl 1-1- (cis, cis-9 ', 1-2'-octadecadienoxy) propane or "CpLinDMA", N, N-dimethyl-3 , 4-dioleyloxybenzylamine or "DMOBA", 1,2-N, N'-dioleylcarbamil-3-dimethylaminopropane or "DOcarbDAP", 2,3-Dylinoleoyloxy-N, N-dimethylpropylamine or "DLinDAP", 1,2-N, N'-Dilinoleilcarbamil-3-dimethylaminopropane or "DLincarbDAP", 1,2-Dilinoleoycarbamil-3-dimethylaminopropane or "DLinCDAP", 2,2-Dilinoleil-4-dim ethyla- [1,3] -dioxolane or "DLin-K-DMA", 2.2-dilinoleyl-4-dimethylaminoethyl- [1,3] -dioxolane or "DLin-K-XTC2-DMA", and 2- (2, 2-di ((9z, 12Z) -octadeca-9,12-dien-1-yl) -1,3- dioxolan-4-yl) -N, N-dimethylethanamine (DLin-KC2-DMA)) (See, WO 2010 / 042877; Semple et al., Nature Biotech.

28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV, et al., Nat. Biotechnol. 23 (8): 1003-1007 (2005); PCT Publication WO2005 / 121348A1).

[0058] The use of cationic cholesterol-based lipids is also contemplated by the present invention. Such cationic lipids in cholesterol can be used alone or in combination with other cationic or non-cationic lipids. Cationic lipids based on cholesterol were included, for example, DC-Chol (N, N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis (3-N-oleylamino-propyl) piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechiques 23, 139 (1997); United States Patent No. 5,744,335), or ICE.

[0059] Cationic lipids, such as dialkylamino-based, imanthane and guanidinium-based lipids are also contemplated. For example, certain parts are directed to a composition comprising one or more cationic lipids based on imidazole, for example, the imidazole cholesterol ester or the lipid "ICE" (3S, 10R, 13R, 17R) -10, 13-dimethyl -17 - ((R) -6-methylheptan-2-yl) -2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [a] phenanthren-3-yl 3- (1 H-imidazol-4-yl) propanoate, as depicted in structure (I) below. In a preferred embodiment, a transfer vehicle for mRNA administration may comprise one or more imidazole-based cationic lipids, for example, the imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R) -10, 13-dimethyl-17 - ((R) -6-methylheptan-2-yl) -2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro- 1H-cyclopenta [a] phenanthren-3-yl 3- (1 H-imidazol-4-yl) propanoate, as represented by structure (I).

Without wanting to be limited by any particular theory, the fusogenicity of the cationic lipid ICE based on imidazole will be created, it will be related to the endosomal break that will be facilitated in the imidazole group, which has a lower pKa in relation to cationic lipids. Endosomal rupture in turn promotes osmotic inflammation and rupture of the liposomal membrane, as well as intracellular transfer of nucleic acid contents (s) loaded into the target cell.

[0060] Imidazole-based cationic lipids are also characterized by their reduced toxicity relative to other cationic lipids. Cationic lipids based on imidazole (eg, ICE) can be combined with traditional cationic lipids, non-cationic lipids and PEG-modified lipids. The cationic lipid may comprise a molar ratio of about 1% to about 90%, about 2% to about 70%, about 5% to about 50%, about 10% to about 40% of the total lipids present in the transfer vehicle , or preferably from about 20% to about 70% of the total lipids present in the transfer vehicle.

[0061] Similarly, certain embodiments are directed to lipid nanoparticles comprising the cationic lipid HGT4003 2 - ((2,3-bis ((9Z, 12Z) -octadeca-9,12-dien-1-yloxy) propyl) disulfanyl) -N, N-dimethylethanamine, as depicted in structure (II) below, and as described in US Provisional Application. N °: 61 / 494,745, filed on June 8, 2011:

[0062] In other embodiments, the compositions described herein are directed to lipid nanoparticles comprising one or more lipid cleavable, such as, for example, one or more cationic lipids or compounds comprising a cleavable disulfide functional group (SS ) (for example, HGT4001, HGT4002, HGT4003, HGT4004 and HGT4005), as described in US Provisional Application. N °: 61 / 494,745.

[0063] Modified polyethylene glycol (PEG) phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl sphingosine-1- [succinyl (methoxy polyethylene glycol) -2000] (C8 PEG-2000 ceramide) were used according to the invention, that is, a lipid nanoparticle. PEG-modified lipids contemplated include, but are not limited to a polyethylene glycol chain of up to 5 kDa in length covalently linked to a lipid with C6-C20 alkyl chain (s) in length. The addition of such components can prevent aggregation and can also be a means to increase the life of the circulation and increase the supply of the composition of the lipid-nucleic acid to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they can be selected to quickly swap out of the formulation in vivo (see US Patent No. 5,885,613). Interchangeable lipids for use are PEG-ceramides that have shorter acyl chains (for example, C14 or C18). The PEG-modified phospholipid and lipids derived from the present invention can be a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% at approximately 10%, approximately 2% of the total lipids present in the liposomal transfer vehicle.

[0064] The present invention also uses non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a series of lipid species that carry a net negative charge at a selected pH, such as physiological pH. The non-cationic lipids include, but are not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoiloleoilfosfatidilcolina (POPC), palmitoyloleoyl-phosphatidylethanolamine ( POPE), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimiristoylphosphoethanolamine (DMPE), distearoyl phosphatidiethanolamine (SOPE), a mixture thereof. Such non-cationic lipids are used in combination with the other excipients, including cationic lipids. Used in combination with a cationic lipid, the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10% to about 70% of the total lipids present in the transfer vehicle.

[0065] Preferably, the transfer vehicle (ie, a lipid nanoparticle) is prepared by combining multiple lipid components (and optionally polymers). For example, a transfer vehicle can be ^{prepared using C12-200,} dOpE, ^{chol, DMG-PEG2K in a molar ratio of 40: 30: 25: 5, or DODAP,} DoPE, cholesterol, DMG-PEG2K in a molar ratio of 18 : 56: 20: 6, or HGT5000, DOPE, chol, DMG-PEG2K in a molar ratio of 40: 20: 35: 5, or HGT5001, DOPE, chol, DMG-PEG2K in a molar ratio of 40:20:35 :5. The selection of cationic lipids, non-cationic lipids and PEG modified lipids that the lipid nanoparticle maintains, as well as the relative molar ratio of said lipids to each other, is based on the characteristics of the selected lipids, the nature of the desired target cells, and the characteristics of the mRNA to be administered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipids. Therefore, molar ratios can be adjusted as a function. For example, to say, the percentage of cationic lipid in the lipid nanoparticle can be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60% or greater than 70 %. The percentage of non-cationic lipids in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30% or greater than 40%. The percentage of cholesterol in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30% or greater than 40%. The percentage of lipids modified with PEG in the lipid nanoparticle may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.

[0066] In certain preferred parts, lipid nanoparticles in the same manner are reduced to future cationic lipids: C12-200, Dlin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In embodiments, the transfer vehicle comprises cholesterol and a PEG modified lipid. On some occasions, transfer vehicles comprise DMG-PEG2K. In certain embodiments, the transfer vehicle comprises one of the following lipid formulations: C12-200, DOPE, col, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, col, DMG-PEG2K, HGT5001, DOPE, col, DMG-PEG2K.

[0067] Liposomal transfer vehicles for use in the compositions of the invention can be prepared by various techniques that are currently known in the art. Multilamellar vesicles (MLV) can be prepared by techniques, for example, depositing a selected lipid on the inner wall of a suitable container or container by dissolving the lipid in a suitable solvent, and then evaporating the solvent to leave a thin film inside the interior. container or by spray drying. Then, a phase can be added to the container with a video movement that results in the formation of MLV. The unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0068] In certain embodiments of this description, the compositions of the present invention comprise a transfer vehicle in which the mRNA is associated both on the surface of the transfer vehicle and encapsulated within the same transfer vehicle. For example, during the preparation of the compositions, cationic liposomal transfer vehicles can be associated with mRNA through electrostatic interactions.

[0069] In certain embodiments, the compositions of the invention can be loaded with diagnostic radionuclides, fluoroscent materials or other materials that are detectable in applications both in vitro and in vivo. For example, diagnostic materials suitable for use in the present invention may include rhodamine dioleoyl phosphatidylethanolamine (Rh-PE), green fluorescent protein mRNA (GFP mRNA), Renilla luciferase mRNA and firefly luciferase mRNA.

[0070] The selection of the appropriate size of a liposomal transfer vehicle must take into account the site of the target cell or tissue and, to some extent, the application for which the liposome is being performed. In some embodiments, it may be desirable to limit mRNA transfection to certain cells or tissues. A liposomal transfer vehicle can be sized so that the dimensions of the liposome have a diameter sufficient to expressly limit or prevent distribution in certain cells or tissues. In general, the size of the transfer vehicle is within the range of about 25 to 250 nm, preferably less than about 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or 10 nm .

[0071] A variety of alternative methods known in the art are available to size a population of liposomal transfer vehicles. One such dimensioning method is described in US Pat. No. 4,737,323. The sonication of a liposome suspension, either by bath or probe, produces a progressive reduction in size to a small ULV of less than about 0.05 microns in diameter. Homogenization is another method that relies on shear energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, the MLVs are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomal vesicles can be determined by near-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10: 421-450 (1981). The average diameter of the liposomes can be reduced by sonication of the formed liposomes. Intermittent ultrasound cycles can be alternated with the QELS evaluation to guide efficient liposome synthesis.

Target cells

[0072] As used herein, the term "target cell" refers to a cell or tissue to which a composition of the invention is directed. In some embodiments, the target cells are deficient in a protein or enzyme of interest.

[0073] In one embodiment, the compositions of the invention facilitate the endogenous production of a subject of one or more functional proteins and / or enzymes, and in particular the production of proteins and / or enzymes that demonstrate less immunogenicity in relation to their counterparts. recombinantly prepared Transfer vehicles comprise mRNA encoding a deficient protein or enzyme. Upon distribution of said compositions to the target tissues and subsequent transfection of said target cells, the exogenous mRNA loaded in the liposomal transfer vehicle (i.e., a lipid nanoparticle) can be translated in vivo to produce a functional protein or enzyme encoded by mRNA administered exogenously (for example, a protein or enzyme in which the subject is deficient). Therefore, the compositions of the present invention will exploit the ability of a subject to use mRNA prepared exogenously or recombinantly to produce a translated protein or enzyme in an endogenous manner, and thereby produce (and when applicable excrete ) a functional protein or enzyme. Proteins or enzymes are translated can also be characterized by the in vivo inclusion of native post-translator modifications that are often absent in recombinantly prepared proteins or enzymes, which further reduces the immunogenicity of the translated protein or enzyme.

[0074] The administration of mRNA encoding a protein or deficient enzyme avoids the need to administer nucleic acids to specific organelles within a target cell (eg, mitochondria). Rather, the transfer of a target cell and the delivery of nucleic acids to the cytoplasm of the target cell, the contents of the mRNA of a transfer vehicle can be translated and a functional protein or enzyme expressed.

[0075] As provided herein, the composition may also comprise the ability to improve the composition of the target cell. Targeted ligands can bind to the outer bilayer of the lipid particle during formulation or postformulation. These methods are well known in the art. In addition, some lipid departmental formulations may employ fusogenic polymers such as PEAA, hemaggluttinin, other lipopeptides (see US Patent Application Numbers 08 / 835,281 and 60 / 083,294) and other features useful for in vivo administration and / or intracellular In other embodiments, the compositions of the present invention demonstrate improved transfection efficiencies, and / or demonstrate improved selectivity towards the target cells or tissues of interest. Therefore, one or more ligands (e.g., peptides, aptamers, oligonucleotides, one vitamin or another) that are capable of improving their affinity and nucleic acid contents for cells can be seen or target tissues. The ligands can be optionally bound or linked to the surface of the transfer vehicle. In some embodiments, the target ligand may encompass the surface of a transfer vehicle or be encapsulated within the transfer vehicle. The ligands are adapted and selected based on their physical, chemical and biological properties (for example, selective affinity and / or recognition of the markers of the target cell surface characteristics). Cell-specific target sites and their corresponding target ligand can vary widely. Directional ligands are selected so that the characteristics of a target cell are exploited, which allows the composition to differentiate between the target and non-target cells. The presentation of said directed ligands that have been conjugated to the moieties present in the transfer vehicle (i.e., a lipid nanoparticle) therefore facilitate the recognition and uptake of the characteristics of the present invention in cells and cells. target tissues. Examples of directed ligands become one or more peptides, proteins, aptamers, vitamins and oligonucleotides.

Application and Administration

[0076] As used herein, the term "subject" refers to any animal (eg, a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, to that the compositions of the invention are administered. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0077] The compositions of the invention provide mRNA administration to treat a number of disorders. The compositions of the present invention are suitable for the treatment of diseases or disorders related to the deficiency of proteins and / or enzymes that are excreted or secreted by the target cell to the surrounding extracellular fluid (e.g., mRNA encoding hormones and neurotransmitters ). In the case, the disease may involve a defect or deficiency in a secreted protein (for example, Fabry disease, or ALS). The symptoms of a disease can be improved in the compositions of the invention (for example, cystic fibrosis). Huntington's health disorders; Parkinson's disease; muscular dystrophies (such as Duchenne and Becker); hemophilia diseases (such as, for example, hemophilia B (FIX), hemophilia A (FVIII), spinal muscle atrophy related to SMN1 (SMA), amyotrophic lateral sclerosis (ALS), galactosemia with GALT, cystic fibrosis (CF); disorders related to SLC3A1 including cystinuria; cancer-related disorders; galactocerebrosidase deficiencies; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis; MPS Sanfilippo syndrome (MPS IIIB); CTNS-related cystinosis; FMR1-related disorders that include fragile X syndrome, tremor / ataxia syndrome associated with fragile X and premature ovarian insufficiency syndrome of fragile X; Prader-Willi syndrome; telangiectasia Hereditary hemorrhagic disease (AT); Niemann-Pick type C1 disease; Diseases related to neuronal zeroide lipofuscinosis, inc sporting juvenile neuronal zeroide lipofuscinosis (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease and PTT-1 and TPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, Childhood ataxia related to EIF2B4 and EIF2B5 with hypomyelination / disappearance of white matter from the central nervous system; Type 2 episodic ataxia is related to CACNA1A and CACNB4; MECP2-related disorders, including classical Rett syndrome, severe neonatal encephalopathy related to MECP2, and PPM-X syndrome; Atypical Rett syndrome related to CDKL5; Kennedy disease (SBMA); Autosomal dominant cerebral arteriopathy related to Notch-3 with subcortical infarcts and leukoencephalopathy (CADASIL); seizure disorders related to SCN1A and SCN1B; Polymerase G-related disorders, which include Alpers-Huttenlocher syndrome, sensory ataxic neuropathy associated with POLG, dysarthria and ophthalmoparesis, and autosomal dominant and recessive progressive external ophthalmopoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Wilson's disease; and Fabry's disease. The mRNA of the invention can encode functional proteins or enzymes that are secreted in the extracellular space. For example, secreted proteins include coagulation factors, complement pathway components, cytokines, chemokines, chemoattractants, protein hormones (e.g., EGF, PDF), whey protein components, antibodies, secretable receptors, and others. In some embodiments, the compositions of the present invention may include mRNA encoding erythropoietin, a1-antitrypsin, carboxypeptidase N or human growth hormone.

[0078] In embodiments, the invention encodes a secreted protein that is formed by subunits that are encoded by more than one gene. For example, the secreted protein may be a heterodimer, wherein each chain or subunit is encoded by a separate gene. It is possible that more than one mRNA molecule is administered in the transfer vehicle and the mRNA encodes a separate subunit of the secreted protein. Alternatively, a single mRNA can be designed to encode more than one subunit (for example, in the case of a single chain Fv antibody). In certain embodiments, separate mRNA molecules encoding individual subunits can be administered in separate transfer vehicles. In one embodiment, the mRNA can encode full-length antibodies (both heavy and light chains of the variable and constant regions) or antibody fragments (e.g., Fab, Fv, or a single Fv chain (scFv) to confer immunity to a In other embodiments, the secreted protein is a cytokine or other secreted protein that comprises more than one subunit (eg, IL-12 or IL-23).

[0079] The compositions of the invention can be administered to a subject. In some embodiments, the composition is formulated in combination with one or more nucleic acids, carriers, targeted ligands or additional stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. For example, in one embodiment, the compositions of the invention can be prepared to deliver mRNA encoding two or more different proteins or enzymes. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pennsylvania, latest edition. The compositions of the present invention can be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the schedule of administration, the age of the subject, sex, the body weight and other relevant factors for doctors of ordinary skill in the art. The "effective amount" for the purposes of this document can be determined by the relevant considerations known to experts in experimental clinical practice, research, pharmacological, clinical and medical techniques. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators that are selected as appropriate measures of disease progression, regression or improvement by those skilled in the art. For example, an adequate amount and a dosage regimen is one that causes at least the production of transient proteins. Aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal or bronchial administration).

[0080] In one embodiment, the compositions of the invention are formulated so as to be suitable for prolonged release of the mRNA contained therein. Said extended release compositions may conveniently be administered to a subject at prolonged dosage intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice daily, daily or every other day. In a preferred embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every two months, every three months, every four months, every six months, every eight months, every nine months or annually. Preferably, the extended release means employed are combined with the modifications made to the mRNA to improve stability.

[0081] Lyophilized pharmaceutical compositions comprising one or more of the liposomal nanoparticles described herein and related methods for the use of such lyophilized compositions as described for example in US Provisional Application are also contemplated herein. No. 61 / 494,882, filed June 8, 2011. For example, lyophilized pharmaceutical compositions according to the invention can be reconstituted prior to administration or reconstituted in vivo. For example, a lyophilized pharmaceutical composition of the disclosure can be formulated in an appropriate dosage form and administered so that the dosage form is rehydrated over time in vivo by the individual's body fluids.

[0082] While certain compounds and compositions of the present invention have been specifically described in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.

[0083] Articles "a" and "a", as used herein, unless clearly stated otherwise, should be understood as plural referents. Descriptions that include "or" between one or more members of a group are considered fulfilled if one, more than one, or all members of the group are present, employed or otherwise relevant to a given product or process, unless otherwise indicated or otherwise evident from the context. When the elements are presented as lists (for example, in the Markush group or in a similar format), it should be understood that each subgroup of the elements is also revealed, and any element can be removed from the group. It should be understood that, in general, when reference is made to the invention, or aspects of the invention, which comprise particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of such elements. , characteristics, etc. For simplicity, these embodiments have not been specifically stated in each case in so many words in this document.

EXAMPLES

Comparative Example 1: Deposit of protein production by intravenous administration of polynucleotide compositions

Messenger RNA

[0084] Human erythropoietin (EPO) (SEQ ID NO: 3; FIG. 3) , human alpha-galactosidase (GLA) (SEQ ID NO: 4;

FIG. 4) , human alpha-1 antitrypsin (A1AT) (SEQ ID NO: 5; FlG. 5) and human factor IX (FIX) (SEQ ID NO: 6; FIG.

6) were synthesized by in vitro transcription of a plasmid DNA template encoding the gene, which was followed by the addition of a 5 'cap structure (Cap1) (Fechter & Brownlee, J. Gen. Virology 86: 1239- 1249 (2005)) and a 3 'poly (A) tail approximately 200 nucleotides in length, as determined by gel electrophoresis. The 5 'and 3' untranslated regions are present in each mRNA product in the following examples and are defined by SEQ ID NO: 1 and 2 ( FlG. 1 and FlG. 2) respectively.

Lipid nanoparticle formulations.

[0085] Formulation 1: 50 mg / ml aliquots of ethanol solutions of C12-200, DOPE, Col and DMG-PEG2K (40: 30: 25: 5) were mixed and diluted with ethanol for a final volume of 3 ml . Separately, a buffered aqueous solution (10 mM citrate / 150 mM NaCl, pH 4.5) of mRNA was prepared from a 1 mg / ml reservoir. The lipid solution was rapidly injected into the mRNA solution so that a final suspension in 20% ethanol does not occur. The nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

[0086] Formulation 2: 50 mg / ml aliquots of ethanol solutions of DODAP, DOPE, cholesterol and DMG-PEG2K (18: 56: 20: 6) were mixed and diluted with ethanol for final volume of 3 ml. Separately, a buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of EPO mRNA was prepared from a 1 mg / ml reservoir. The lipid solution was rapidly injected into the mRNA solution so that a final suspension in 20% ethanol does not occur. The nanoparticle suspension is filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C. Final concentration = 1.35 mg / ml EPO mRNA (encapsulated). Z ave - 75.9 nm (Dv (50) - 57.3 n ^ n> Dv (90) - 92.1 nm).

[0087] Formulation 3: 50 mg / ml aliquots of ethanolic solutions of HGT4003, DOPE, cholesterol and DMG-PEG2K (50: 25: 20: 5) were mixed and diluted with ethanol for final volume of 3 ml. Separately, a buffered aqueous solution (10 mM citrate / 150 mM NaCl, pH 4.5) of mRNA was prepared from a 1 mg / ml reservoir. The lipid solution was rapidly injected into the mRNA solution so that a final suspension in 20% ethanol does not occur. The nanoparticle suspension is filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

[0088] Formulation 4: 50 mg / ml aliquots of ethanol solutions of ICE, DOPE and DMG-PEG2K (70: 25: 5) were mixed and diluted with ethanol for final volume of 3 ml. Separately, a buffered aqueous solution (10 mM citrate / 150 mM NaCl, pH 4.5) of mRNA was prepared from a 1 mg / ml reservoir. The lipid solution was rapidly injected into the mRNA solution so that a final suspension in 20% ethanol does not occur. The nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

[0089] Formulation 5: 50 mg / ml aliquots of ethanolic solutions of HGT5000, DOPE, cholesterol and DMG-PEG2K (40: 20: 35: 5) were mixed and diluted with ethanol for final volume of 3 ml. Separately, a buffer solution (10 mM citrate / 150 mM NaCl, pH 4.5) of EPO mRNA was prepared from a stock of 1 mg / ml. The lipid solution was rapidly injected into the mRNA solution so that a final suspension in 20% ethanol does not occur. The nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C. Final concentration - 1.82 mg / ml EPO mRNA (encapsulated). Zave - 105.6 nm (Dv (50) - 53.7 nm; Dv (90) - 157 nm).

[0090] Formulation 6: Aliquots of 50 mg / ml ethanolic solutions of HGT5001, DOPE, cholesterol and DMG-PEG2K (40: 20: 35: 5) were mixed and diluted with ethanol to a final volume of 3 ml. Separately, a buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of EPO mRNA was prepared from a 1 mg / ml reservoir. The lipid solution was rapidly injected into the mRNA solution so that a final suspension in 20% ethanol does not occur. The nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

Analysis of the protein produced through an injection protocol of nanoparticles loaded with mRNA administered intravenously

[0091] Studies were conducted using male CD-1 mice approximately 6-8 weeks of age at the beginning of each experiment, unless otherwise indicated. Samples were introduced by a single injection into the tail vein for a total dose equivalent to 30-200 micrograms of encapsulated mRNA. The mice were sacrificed and perfused with the saline solution at the designated time points.

Isolation of organic tissues for analysis.

[0092] The liver and spleen of each mouse was harvested, divided into three parts, and stored in any of 10% of neutral buffered formalin or complement frozen and stored at -80 ° C for analysis.

Isolation of serum for analysis.

[0093] All animals were sacrificed for CO2 asphyxiation 48 hours after post dose administration (± 5%) followed by thoracotomy and collection of terminal cardiac blood. Whole blood was collected (maximum volume obtainable) by means of cardiac puncture in animals sacrificed in serum separator tubes, which are allowed to coagulate at room temperature for less than 30 minutes, focused at 22 ° C ± 5 ° C at 9300 g for 10 minutes, and serum extracted. For provisional blood collection, approximately 40-50 g of whole blood is recognized by puncture of the facial vein or tail cut. Samples of untreated animals are used as a reference for comparison with study animals.

Enzyme-linked immunosorbent assay analysis ( ELISA)

[0094] EPO ELISA: Quantification of the EPO protein was performed following the procedures reported for the human EPO ELISA kit (Quantikine IVD, R&D Systems, Catalog No. Dep-00). The positive controls used consisted of recombinant human erythropoietin protein and tissue culture grade (R&D Systems, catalog number 286-EP and 287-TC, respectively). Detection is controlled by absorption (450 nm) in a Molecular Device Flex Station instrument.

[0095] GLA ELISA: standard ELISA procedures were used using sheep anti-alpha-galactosidase G-188 IgG as a capture antibody with rabbit anti-alpha-galactosidase TK-88 IgG as the secondary (detection) antibody ( Shire Human Genetic Therapies). Goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) was used for activation of the 3,3 ', 5,5'-tetramethylbenzidine (TMB) substrate solution. The reaction was quenched using 2N H2SO4 after 20 minutes. Detection is controlled by absorption (450 nm) in a Molecular Device Flex Station instrument. Negative and positive media were used, respectively.

[0096] FIX ELISA: Quantification of the FIX protein was performed following the procedures reported for the human FIX ELISA kit (AssayMax, Pro Assay, Catalog # EF1009-1). A1ATELISA: The quantification of the A1AT protein was performed following the procedures reported for the human A1AT ELISA kit (Innovative Research, Catalog N2 IRAPKT015).

Western blot analysis

[0097] ( EPO): Western blot analysis was performed using an anti-hEPO antibody (R&D Systems No. MAB2871) and ultrapure human EPO protein (R&D Systems No. 286-EP) as a control.

Results

[0098] The work described in this example demonstrates the use of lipid nanoparticles encapsulated with mRNA as a reservoir source for protein production. Said deposition effect can be achieved at multiple sites within the body (ie, liver, kidney, spleen and muscle). Measurement of the desired exogenous protein derived from messenger RNA administered through liposomal nanoparticles was achieved and quantified, and protein secretion from a deposit with human erythropoietin (hEPO), human alpha-galactosidase (hGLA), human alpha-1 antitrypsin (hA1AT), and the human iX factor mRNA (hFIX) was demonstrated.

1A. Results of in vivo protein production of human EPO

[0099] The production of hEPO protein was demonstrated with various formulations of lipid nanoparticles. Of four different cationic lipid systems, C12-200-based lipid nanoparticles produced the greatest amount of hEPO protein after four hours after intravenous administration as measured by ELISA ( FIG. 7 ). This formulation ( Formulation 1) resulted in 18.3 ug / mL of hEPO protein secreted in the bloodstream. Normal serum hEPO protein levels for humans are 3.3-16.6 mlU / ml (Document NCCLS C28-P; Vol. 12, No. 2). Based on a specific activity of 120,000 Ul / mg of EPO protein, which produces an amount of 27.5-138 pg / mL of hEPO protein in normal human individuals. Therefore, a single dose of 30 gg of a C12-200-based cationic lipid formulation encapsulating hEPO mRNA produced an increase in the respective protein of physiological levels more than 100,000 times.

[0100] Of the lipid systems tested, the formulation of lipid nanoparticles based on DODAP was the least effective. However, the observed amount of human EPO protein derived from delivery through a DODAP-based lipid nanoparticle that encapsulates EPO mRNA was 4.1 ng / ml, which is still more than 30 times higher than physiological levels. normal EPO protein ( Table 1) .

Table 1 Crude values of secreted HEPO protein for various lipid-based cationic nanoparticle systems as measured by ELISA analysis (as shown in FIG. 8) . Doses are based on encapsulated hEPO mRNA. Protein values are represented as nanograms of human EPO protein per milliliter of serum. Changes in hematocrit are based on the comparison of previous bleeding (Day -1) and Day

10.

[0101] In addition, the resulting protein was tested to determine if it was active and functioned correctly. In the case of mRNA replacement therapy (MRT) that uses hEPO mRNA, changes in hematocrit are monitored over a period of ten days for five different lipid nanoparticle formulations ( Figure 8, Table 1) to evaluate the protein activity During this period of time, two of the five formulations demonstrated the hematocrit increase (> 15%), which is indicative of the production of the active hEPO protein from these systems.

[0102] In another experiment, hematocrit changes were monitored over a period of 15 days ( FIG. 9, Table 2) . The lipid nanoparticle formulation ( Formulation 1) was administered as a single dose of 30 pg, or as three small doses of 10 pg injected on day 1, day 3 and day 5. Similarly, Formulation 2 was administered as 3 doses of 50 (pg on day 1, day 3 and day 5. C12-200 produced a significant increase in hematocrit. In general, an increase in change of up to ~ 25% was observed, which is indicative of the protein activates human EPO that is produced from such systems.

Table 2 Hematocrit levels of each group during an observation period of 15 days ( FIG. 9) . The mice were given a single injection or three injections every two days. N = 4 mice per group.

1 B. Results of the production of human GLA protein in vivo

[0103] A second exogenous-based protein system was explored to demonstrate the "deposition effect" when using mRNA-loaded lipid nanoparticles. Animals were injected intravenously with a single dose of 30 micrograms of encapsulated human alpha-galactosidase mRNA (hGLA) using a lipid nanoparticle system based on C12-200 and sacrificed after six hours ( Formulation 1). The quantification of the secreted hGLA protein was performed by ELISA. Untreated mouse serum and human alpha-galactosidase protein were used as controls. The detection of the human alpha-galactosidase protein was monitored for a period of 48 hours.

[0104] Measurable levels of hGLA protein were observed throughout the entire time course of the experiment with a maximum level of 2.0 ug / ml of hGLA protein at six hours ( FIG. 10 ). Table 3 lists the specific amounts of hGLA found in the serum. Normal activity in healthy human men has been reported to be approximately 3.05 nanomol / h / mL. The activity of alpha-galactosidase, a recombinant protein of human alphagalactosidase, 3.56 x 106 nanomol / h / mg. The analysis of these values yields an amount of approximately 856 pg / ml of hGLA protein in normal healthy male individuals. The amount of 2.0 ug / ml hGLA protein observed after six hours when a lipid nanoparticle loaded with hGLA mRNA is administered is more than 2,300 times greater than normal physiological levels. In addition, after 48 hours, appreciable levels of hGLA protein (86.2 ng / mL) can still be detected. This level is representative of almost 100 times larger amounts of hGLA protein over physiological amounts still present at 48 hours.

Table 3. Crude values of hGLA protein secreted over time as measured through ELISA (as shown in FIG. 10) . Values are represented as nanograms of hGLA protein per milliliter of serum. N = 4 mice per group.

[0105] In addition, the half-life of alpha-galactosidase when administered at 0.2 mg / kg is approximately 108 minutes. The production of GLA protein through "deposition effect" when lipid nanoparticles loaded with GLA mRNA are administered shows a substantial increase in residence time in the blood compared to direct injection of the naked recombinant protein. As described above, significant amounts of protein are present after 48 hours.

[0106] The Activity Profile of a-galactosidase protein produced from lipid nanoparticles loaded with GLA mRNA was measured as a function of the metabolism of 4-methylumbelliferyl-aD-galactopyranoside (4-MU-a-gal). As shown in FIG. 11, the protein produced from these nanoparticle systems is quite active and reflects the available protein levels ( FIG. 12, Table 3) . AUC comparisons of hGLA production are based on mRNA therapy versus enzyme replacement therapy (ERT) in mice and humans at an increase of 182 and 30 times, respectively (Table 4 ).

Table 4. Comparison of the Cmax and AUCinf values in patients with Fabry after Administration of IV 0.2 mg / kg of alpha-galactosidase (pharmacological dose) with those of mice after administration of IV alphagalactosidase and GLA mRNA. 8 The data was published in a published article (Gregory M. Pastores et al. Safety and Pharmacokinetics of hGLA in patients with Fabry disease and end-stage renal disease. Nephrol Dial T ransplant (2007) 22: 1920-1925. B Kidney disease non-terminal c-a-galactosidase activity at 6 hours after administration.

[0107] The ability of lipid nanoparticles encapsulated by mRNA to target organs that can act as a reservoir for the production of a desired protein has been demonstrated. The levels of secreted protein observed have been several orders of magnitude above normal physiological levels. This "deposit effect" is repeatable. FIG. 12 again shows that robust protein production is observed in the dosage of wild-type mice (CD-1) with a single dose of 30 µg of hGLA loaded with mRNA in lipid nanoparticles based on C12-200 ( Formulation 1). In this experiment, hGLA levels are evaluated over a period of 72 hours. A maximum average of 4.0 ug of human hGLA protein serum / ml of serum was detected six hours after administration. Based on a value of ~ 1 ng / ml hGLA protein for normal physiological levels, hGLA MRT provides approximately 4,000 times higher protein levels. As before, the hGLA protein could be detected up to 48 hours after administration ( FIG. 12) .

[0108] An analysis of isolated tissues from this same experiment provides information on the distribution of hGLA protein in mice treated with MRT hGLA ( FIG. 13) . Supraphysiological levels of the hGLA protein were detected in the liver, spleen and kidneys of all mice with a maximum of between 12 and 24 hours after administration. Detectable levels of MRT-derived protein are observed three days after a single injection of lipid nanoparticles loaded with hGLA.

[0109] In addition, the production of hGLA after administration of C12-200 nanoparticles loaded with hGLA mRNA shows a dose response in the serum ( FIG. 14A) , as well as in the liver ( FIG. 14B) .

[0110] An inherent feature of lipid-mediated nanoparticle RNA replacement therapy would be the pharmacokinetic profile of the respective protein produced. For example, ERT based treatment of mice using alpha-galactosidase resulted in a plasma half-life of approximately 100 minutes. In contrast, MRT-derived alpha-galactosidase has a residence time in the blood of approximately 72 hours with a maximum time of 6 hours. This means a much greater exposure for the organs to participate in a possible continuous uptake of the desired protein. A comparison of the PK profiles is shown in FIG.

15 and demonstrates the large difference in cleaning rates and, ultimately, a significant change in the area under the curve (AUC) can be achieved through MRT-based treatment.

[0111] In a separate experiment, hGLA MRT was applied to a mouse disease model, the KO hGLA mice (Fabry mice). A dose of 0.33 mg / kg of C12-200-based lipid nanoparticles loaded with hGLA mRNA ( Formulation 1) was administered to female KO mice as a single intravenous injection. Substantial amounts of MRT-derived hGLA protein were produced with peak at 6 hours (~ 560 ng / ml of serum) which is approximately 600 times higher than normal physiological levels. In addition, the hGLA protein was still detectable 72 h after administration ( FIG. 16) .

[0112] The quantification of the GLA protein derived from MRT in vital organs demonstrated substantial accumulation as shown in FIG. 17. A comparison of the MRT-derived hGLA protein observed with the normal physiological levels found in the key organs is shown (normal levels are represented as dashed lines). While protein levels at 24 hours are higher than at 72 hours after administration, the hGLA protein levels detected in the liver, kidney, spleen and hearts of treated Fabry mice are equivalent to those wild type levels. For example, 3.1 ng of hGLA protein / mg of tissue have been found in the kidneys of treated mice 3 days after a single MRT treatment.

[0113] In a subsequent experiment, a comparison of ERT-based alpha-galactosidase treatment versus hGLA MRT-based treatment of male Fabry KO mice was made. A single intravenous dose of 1.0 mg / kg was administered for each therapy and the mice were sacrificed one week after administration. Serum hGLA protein levels are monitored at 6 hours and 1 week after injection. The liver, kidney, spleen and heart will be analyzed to determine the accumulation of hGLA protein one week after administration. In addition to biodistribution analyzes, a measure of efficacy was determined through the measurement of globotrioasilceramide (Gb3) and lyso-Gb3 reductions in the kidney and heart. FIG. 18 shows serum hGLA protein levels after treatment of lipid nanoparticles loaded with alphagalactosidase mRNA or GLA ( Formulation 1) in male Fabry mice. Serum samples will be analyzed at 6 hours and 1 week after administration. A robust signal was detected for mice treated with MRT after 6 hours, with serum levels of hGLA protein of ~ 4.0 ug / ml. In contrast, there was no detectable alpha-galactosidase in the bloodstream at this time.

[0114] Fabry mice in this experiment were sacrificed one week after the initial injection and the organs were recognized and analyzed (liver, kidney, spleen, heart). FIG. 19 shows a comparison of the human GLA protein found in each respective organ after treatment with hGLA MRT or ERT alphagalactosidase. The levels correspond to an hGLA present one week after administration. The hGLA protein was detected in all the organs analyzed. For example, mice with MRT resulted in an accumulation of hGLA protein in the kidney of 2.42 ng protein hGLA / mg protein, while mice with alpha-galactosidase have only residual levels (0.37 ng / mg of protein). This corresponds to a ~ 6.5 times higher level of hGLA protein when treated through MRT hGLA. Following the analysis of the heart, 11.5 ng of hGLA protein / mg protein for the MRT-treated cohort was found compared to only 1.0 ng / mg of alpha-galactosidase protein. This corresponds to an accumulation ~ 11 times higher in the heart for mice treated with hGLA MRT on ERT-based therapies.

[0115] In addition to the biodistribution analyzes performed, efficacy evaluations are determined by measuring globotrioasilceramide (Gb3) and lyso-Gb3 levels in key organs. A direct comparison of the reduction of Gb3 after a single intravenous treatment of 1.0 mg / kg. GLA MRT is a considerable difference in Gb3 levels in the kidneys and in the heart. For example, Gb3 levels for GLA MRT vs. alpha-galactosidase produced reductions of 60.2% versus 26.8%, respectively ( FIG. 20) . In addition, Gb3 levels in the heart were reduced by 92.1% versus 66.9% for MRT and alpha-galactosidase, respectively ( FIG. 21 ).

[0116] A second biomarker relevant for measuring efficacy is smooth-Gb3. GLA MRT reduced lyso-Gb3 more effectively than alpha-galactosidase also in the kidneys and heart ( FIG. 20 and FIG. 21, respectively). In particular, Fabry mice treated with MRT demonstrated smooth-Gb3 reductions of 86.1% and 87.9% in the kidneys and heart compared to mice with alpha-galactosidase, which will result in a decrease of 47.8 % and 61.3%, respectively.

[0117] The results with lipid nanoparticles for hGLA in C12-200 are based on other formulations of lipid nanoparticles. For example, hGLA mRNA loaded in HGT4003 ( Formulation 3) or lipid nanoparticles based on HGT5000 ( Formulation 5) administered as a single IV dose as a result of hGLA production at 24 hours after administration ( FIG. 22 ) . HGLA production exhibited a dose response. Similarly, hGLA production refers to 6 hours and 24 hours after

Claims (14)

1. A composition comprising (a) at least one mRNA molecule at least a portion of which encodes a functional secreted polypeptide; and (b) a liposomal transfer vehicle comprising a lipid nanoparticle comprising one or more cationic lipids, one or more non-cationic lipids and one or more lipids modified with PEG to use a method of treating a subject having a defect or deficiency in said secreted polypeptide, in which the mRNA is encapsulated in the transfer vehicle and in the composition is administered by pulmonary administration by nebulization. 2. The composition for use with claim 1, wherein the defect or deficiency refers to a disease or disorder comprising one of Huntington's disease; Parkinson's disease; muscular dystrophies (such as Duchenne and Becker); hemophilia diseases (such as hemophilia B (FIX), hemophilia A (FVIII); spinal muscular atrophy related to SMN1 (SMA); amyotrophic lateral sclerosis (ALS); galactosemia with GALT; cystic fibrosis (CFC); disorders related to SLC3A1 including cystinuria, disorders related to COL4A5 including Alport syndrome; deficiencies of galactocerebrosidase; adrenoleukodystrophy and adrenomyeloneuropathy linked to the X chromosome, Friedreich ataxia, Pelizaeus-Merzbacher disease; tuberous sclerosis related to TSC1 and TSC2 syndrome; Bfil2 syndrome; (MPS IIIB), CTNS-related cystinosis; FMR1-related disorders that include fragile X syndrome, tremor / ataxia syndrome associated with fragile X and premature ovarian insufficiency syndrome of fragile X; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick type C1 disease; Lipofuscinosis-related diseases Neural zeroides include juvenile zero neuronal lipofuscinosis (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Biels-chowsky disease, and PTT-1 and TPP1 deficiencies; Childhood ataxia related to EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5 with hypomyelination / disappearance of white matter from the central nervous system; Type 2 episodic ataxia is related to CACNA1A and CACNB4; MECP2-related disorders, including classical Rett syndrome, severe neonatal encephalopathy related to MECP2, and PPM-X syndrome; Atypical Rett syndrome related to CDKL5; Kennedy disease (SB-MA); Autosomal dominant cerebral arteriopathy related to Notch-3 with subcortical infarcts and leukoencephalopathy (CADASIL); seizure disorders related to SCN1A and SCN1B; Polymerase related disorders; Alpers-Huttenlocher syndrome, sensory ataxic neuropathy associated with POLG, dysarthria and ophthalmoparesis, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Wilson's disease; and Fabry's disease. 3. The composition for use according to claim 1 or claim 2, wherein the mRNA encodes an enzyme that is abnormally deficient in an individual with a lysosomal storage disorder. 4. The Composition for use according to claim 1 or claim 2, wherein the mRNA encoded polypeptide is erythropoietin, a-galactosidase polypeptide, LDL receptor, factor VIII, factor IX, aL-iduronidase , iduronate sulfatase, heparin-N-sulfatase, aN-acetylglucosaminidase, galactose 6-sulfatase, pgalactosidase, acidic lysosomal lipase or arylsulfatase-A polypeptide. 5. The composition for use according to any one of responses 1-4, wherein the RNA molecule comprises: At least one modification that confers stability to the RNA molecule; a modification of the 5 ’untranslated region of said RNA molecule, optionally in which said modification comprises the inclusion of a Cap1 structure; or a modification of the 3 ’untranslated region of said RNA molecule, optionally wherein said modification comprises the inclusion of a polyA tail. 6. The composition for use according to any one of responses 1-5, which also comprises an agent to facilitate the transfer of the RNA molecule and an intracellular compartment of a target cell, optionally wherein said target cell is selected from the group consisting of hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, mesenchymal stem cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells , pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells. 7. The composition for use according to any one of responses 1-6, wherein the lipid nanoparticle comprises C12-200; DLinKC2DMA, CHOL, DOPE and DMG-PEG-2000; or C12-200, DOPE, CHOL and DMGPEG2K. 8. The composition for use according to any one of responses 1-7, wherein the lipid nanoparticle comprises a cleavable lipid. 9. The composition for use according to any one of responses 1-8, wherein said composition is lyophilized. 10. The composition for use according to any one of responses 1-9, wherein said composition is a reconstituted lyophilized composition. 11. The composition for use according to any one of responses 1-10, wherein the composition is for administration to the subject twice a week, once a week, every ten days, every two weeks or every three weeks. 12. The composition for use according to any one of responses 1-11, in which the size of the transfer vehicle is within the range of about 25 to 250 nm. 13. The composition for use according to any one of responses 1-11, wherein the size of the transfer vehicle is less than 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or 10 nm. 14. The composition for use according to any one of responses 1-13, wherein the polypeptide is expressed at least one therapeutic level for more than one, more than four, more than six, more than 12, more 24, more than 48 hours, or more than 72 hours after administration, optionally, where the level of the protein is detectable at 3 days, 4 days, 5 days or 1 week or more after administration.